Micro indirect hemagglutination test for detection of antibody against transmissible gastroenteritis virus of pigs

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Micro-Indirect Hemagglutination Test for Detection of

Antibody Against Transmissible

Gastroenteritis

Virus

of Pigs

M. SHIMIZU* AND Y. SHIMIZU

Hog Cholera ResearchSection,National Institute ofAnimalHealth, Kodaira, Tokyo, 187, Japan

Received forpublication8April 1977

A micro-indirect hemagglutination (IHA) test was developed for detecting antibody against transmissible gastroenteritis (TGE) virus ofpigs. TGE virus propagated in swine kidney cell cultureswashighly purified and concentrated

by thecombination of ammonium sulfate precipitation, treatment with fluoro-carbon, andsucrosedensity gradient centrifugation. Tanned sheep erythrocytes weresensitized with purified virus foruseinthe IHAtest.The results oftesting

104 serum samples collected from pigs in the field indicated that the IHA

antibody titers wereapproximately five times higher than those obtained by a

serum neutralization test and that there was good correlation between the antibody titers determined by thetwotests. High IHA antibodytitersdeveloped in pigsexperimentally exposedtovirulentTGE virus. Sensitized sheep

erythro-cytes were stable under long-term storage at 4°C (at least for 50 days). The

conclusions made are that the IHA test described is more sensitive than the serumneutralizationtestfor the detection of TGEantibody andmaybe ofvalue forserodiagnosis of TGE.

Becausetransmissible gastroenteritis (TGE)

of pigs is ahighlycontagious viral diseaseand

usually spreads veryrapidly to all susceptible

pigs in aherd, rapidand reliable diagnosis is

required for the control of the disease.

Al-though several serological tests for detecting

TGE antibody have been developed (2, 8, 14, 15), a serum neutralization (SN) test is the

preferred methodforserodiagnosisofTGE,

es-peciallyifserumsamplescollected from pigs in

both acute and convalescent stages of the

dis-ease are tested. The SN test gives reliable re-sults and is of value for thedetection and mea-surementofTGE antibody.

However,performingthe SN test is time

con-suming and requiresfacilities for cell culture. Indirecthemagglutination (IHA), which is a sensitive and rapid serological test, has been applied for the detectionof antibodies against many viruses (4, 9, 10, 13).

Using erythrocytes sensitized with highly

purified and concentrated TGE virus, an at-tempt was made to utilize the IHA test for

detectingand measuring TGE antibody in pig sera.

The purpose of the present report is to de-scribe the development ofthe IHA test for de-tecting and measuring TGE antibody and its

applicability toserodiagnosis ofTGE.

91

MATERIALS AND METHODS

Viruses. The TO strain of an attenuated TGE

virus wasused for the SN test and for production of

the antigen used for sensitizing sheep erythrocytes

(SRBC)inthe IHAtest.The strainhad beenserially

passagedinswinekidney cell cultures (7) and was

used at passage168for these experiments.

TheShizuoka strain (12), a virulent TGE virus,in

the form of a 10% suspension of small intestine of

infected piglet, was used for experimental

inocula-tion ofpigs.

Cell cultures. Monolayers of primary swine

kid-ney cells growninRouxbottles (150 by220mm) and

testtubes (10by100mm)wereused for the

produc-tionofantigenand for the SN test,respectively. The

growth mediumconsisted of Earle balanced salt

so-lution containing 0.5% lactalbumin hydrolysate

(LE) supplemented with 10% heat-inactivated

bo-vine serum, 0.021% sodium bicarbonate, 20 ,tg of

kanamycin perml, and 100

jig

of streptomycin per

ml. The maintenance medium was LE

supple-mented with 3% heat-inactivated bovine serum,

0.07%sodiumbicarbonate, and thesameantibiotics

as in thegrowthmedium.

Antigen for sensitization of SRBC. Confluent

monolayers of swine kidney cells grown in Roux

bottles wereinoculated with 20 ml ofviral

suspen-sionof theTO strain, containing106-0mediantissue

culture infective doses (TCID5e)/ml. The virus was

allowedtoadsorbfor 1 hat37°C, and then80ml of

the maintenance medium wasaddedto the culture

bottles.Thecultures were incubated for 36 h at37°C,

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then culture fluid was harvested and clarified by Sera. Serumsampleswerecollected from 104pigs

low-speed centrifugation at 1,000xg for 10 min, and raised in the field farms. Thepigs had been exposed

the virus was purified and concentrated as follows. to the virulent field strain of TGE virus or

inocu-Solid ammonium sulfate wasaddedtotheharvested lated with the TO-163 vaccine strain (5). All sera

fluidto 45%saturation.Themixturewasstirred for wereinactivated at 56°Cfor 30 min and tested for

3h at4°C and then centrifugedat 1,500 xg for 20 TGE antibody byboth the IHA and SN tests.

min.The precipitate wasresuspendedinphosphate- Exposure of experimental pigs to the virulent

buffered saline (PBS; pH 6.4) to approximately 1/25 TGE virus. Fifteen Yorkshirepigs weighing15to25

of the original volume. The viral suspension was kg, which had beenserologically negativefor TGE

then mixed with one-third volume offluorocarbon antibody, were exposed orally to 103 median pig

(Diflon solvent S3, Daikin Kogyo Co., Osaka, Ja- infective doses of the virulent Shizuoka strain of

pan), shakenvigorously for1 min, andcentrifuged TGE virus. Sera were collected before and after

at 1,000 x g for 5 min. The aqueous phase was exposure and tested for both IHA and SN antibodies.

collectedand centrifugedat56,000 xg for2h.Virus IHA test. Heat-inactivatedsera werediluted 1:5

pellets were resuspended in approximately 1/100 with the diluent medium and adsorbed with 2

vol-volume of PBS. Then, 1 ml of concentrated viral umes of a 50% suspension of Formalin-treated,

suspensionwaslayeredontop ofalinear10to 50% tanned SRBC for 1 hat37°C withoccasional

shak-sucrosedensity gradient preparedin acellulose ni- ing. Thesupernatantfluidswereconsideredto

rep-tratetube andcentrifuged at 85,000 xgfor 3h ina resenta 1:10serumdilution andwereused for the

Spinco SW27.1 rotor. The virus band formed was IHAtest.

collected, dialyzed against three changes of PBS, The test was carried out by the micromethod,

andused for sensitization of SRBC. using plasticmicrotrayswithV-shapedcups. Serial

Preparation of sensitized SRBC. SRBC in 8% twofold dilutions ofsera were made inavolume of

suspensionweremixed withanequalvolume of3% 0.025 ml withmicrodiluters, and 0.025 ml of

sensi-Formalin solutioninPBS, incubatedat370C for18 tized SRBCsuspensionwasaddedtoeach dilution.

h, washed three times with distilled water, and The microtrays were shaken and incubated

over-suspended to a 10% suspension in PBS. The For- nightat40C.Settling patternsof SRBCwerereadas

malin-treated SRBC were treated with an equal 100% agglutination when uniformly agglutinated

volume of0.005%tannicacid solutioninPBSat37°C cells had coated the bottom of the wells. The IHA

for 15 min, washed twice, and suspended in PBS. antibodytiterwasexpressedasthereciprocalof the

TheFormalin-treated, tanned SRBCwerethensen- highestdilution ofserumthatshowed50%

aggluti-sitized with TGEantigenbymixing1volume of10% nation(Fig. 1).

cell suspension and 5 volumes ofa suspension of SNtest.The SNtest wasperformed accordingto

purified TGE virus and incubating at 37°C for 30 the method of Haradaetal. (8). Briefly,serial

two-min. The sensitized cells were washedtwice with folddilutions ofsera were incubated withanequal

diluent mediumconsisting of PBS (pH 7.2)supple- volume of viralsuspension containing200 TCID50/

mented with 0.1% gelatin and1% heat-inactivated 0.1ml of theTO strainat37°Cfor1h.Two tubes of

normal rabbit serum, which had been previously swinekidneycell cultureswereinoculated with0.1

adsorbed with50%Formalin-treated,tanned SRBC. ml of each serum-virus mixture. All cultures

re-Finally, the cells were suspended in the diluent ceived 0.5 ml of maintenance medium and were

medium to a 1% suspension and used in the IHA incubated in aroller drumat370C for4 days. The

test. occurrenceofcytopathiceffectwasdetermined,and

SERUM No. IIlR

1 t te e i

1J»

2

QQ

QQQ(6f3

3

_0

4 00 3 i < ( t ) G

@80

50 E) 3209

6 640

9';;:)()C3}(;9*}V )4 5; * @

2~~~~L560

1110 1/40 1/160 1/640

1/1260

SERUM DILUTION

FIG. 1. Hemagglutinatingpatternsinthe IHA testfor TGEantibody.IHAantibodytitersareindicatedin

therightcolumn.

92

SHIMIZU AND SHIMIZU J.

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theSN antibodytiterwasexpressedasthe

recipro-calofthe highestdilution ofserumthatcompletely

inhibited cytopathic effect in the test.

RESULTS

Virus purification and concentration. The recovery of infectious virus during the purifica-tionandconcentrationprocedures is shown for arepresentative experiment in Table 1. Some loss of infectious virus occurred at each step of the procedure.

The virus wasefficientlyprecipitated by am-monium sulfate (recovery of 78%), as reported

by Garwesand Pocok (6).

Treatment with fluorocarbon did not greatly reduce virus infectivity, indicating that

fluoro-carbon treatment is one of the useful methods

for removing contaminating protein from the crude viral suspension.

However, a great loss of infectious virus

oc-curred with sucrose density gradient

centrifu-gation.Thismaybe due toincomplete recovery of the virus rather than degradation of viral

particles during thecentrifugation.

Fifteen percent of infectious virus were fi-nally recovered in the experiment shown in Table 1.

Determination of optimum concentration of virusforsensitizingSRBC. To examine the effect of the virus concentration used for sensi-tizing SRBC on the IHA antibodytiters, puri-fied virus containing 109 "

TCID50/ml

was

seri-ally dilutedintwofoldsteps andusedto sensi-tizeSRBC. The results of tests on five sera are

shown in Table 2. It is evident that the IHA

antibodytitersobtainedweredependentonthe virus concentration used for sensitizing SRBC. When SRBCcoatedwith 1081'

TCID.50

of virus per ml were used in the test, the IHA

antibodytiters obtained were extremely lower in two (sera 3 and 4) of four positive sera as

comparedwith thoseobtained with morethan 10#4

TCIDjo

of the virus per ml. SRBC sensi-tized with 1078

TCID5e

of the virus per ml gave negative results in three positive sera (sera 2, 3,and4). More consistentresultswereobtained

when the serum samples were tested with

SRBCsensitizedwith more than 1084TCID5, of the virus per ml. Therefore, SRBC sensitized

with virus titering more than 108-

TCID50/ml

were prepared and used in subsequent experi-ments.

Typical IHA patterns are shown in Fig. 1. Relationship between IHA and SNantibody

titers. Seracollectedfrom 104 pigs in thefield weretestedforantibodiesbyboth the IHAand

SN tests to compare the sensitivity of these

testsfordetectingTGE antibody andto exam-inethe correlation betweenantibodytiters

ob-TABLE 1. Recovery of virus during purification and

concentrationprocedures

Vol Infectivity Recovery

Virussample (ml) (TCID55/ml) (%)

Cell culturefluid 5,000 1067 100 Ammoniumsulfatepre- 195 108J 78

cipitate

After treatment with 171 108" 68

fluorocarbon

Pelleted virus by high- 2 10 8 50 speed centrifugation

Virusbandin 10 to50% 3 108'l 15 sucrose density

gra-dient

TABLE 2. Effect of virus concentration used for sensitizingSRBConthe IHA test

IHAantibodytiter Virus concn

(TCID50/ml) Serum Serum Serum Serum Serum

1 2 3 4 5

1090

80 160 320 320 <10

10'7 160 160 160 320 <10

101-4 80 80 160 320 <10

108.' 80 80 40 80 <10

107.S 40 <10 <10 <10 <10

204B8

512

uJ F 128

0

C 32

z

z 8

Ln

2

1>1

* 0 0 s 0

*

rr

t

*

"

r

- * * w

*

rN r

0

* S* - S

.

* 6 S

r=+0.865

-S'S

10> 20 80 320 1280 5'20

IHA ANTIBODY TITER

FIG. 2. Correlation between IHA and SN

anti-bodytiters.

tained by each test (see Fig. 2). The average

antibody titers obtained by the IHA and SN tests were 125 and 25, respectively.

Although with a few exceptions the IHA

antibodytiters werehigher(approximatelyfive times) thanSN antibodytiters, antibodytiters

obtainedbythe two testsshowed

good

correla-tion.Thecorrelationcoefficientwas+0.865and wasstatistically significant (P <0.001).

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Antibody responses of pigs experimentally exposed to virulent TGE virus. Fifteen pigs negative for TGE antibody were orally exposed

to 103-0 median pig infective doses of the viru-lent Shizuoka strain of TGEvirus.

Seraobtained before and after exposure were tested for antibodies by both the IHA and SN tests.

All pigs exposed showed severe diarrhea after incubation periods of 1 to 3 days, which continued for2to4days.

TGE antibody was demonstrated by both

IHAand SN tests in the convalescent sera col-lected from all infectedpigs on days 7 and 14 of

infection (Table 3). Antibodies detectable by both tests gradually increased in titer with the lapse oftime and reached titersof 640 and 128 inthe IHA and SN tests, respectively, 14 days

after exposure. Again, the IHA antibody titers were higher (approximately five times) than those of SN antibodyinthisexperiment.

Stability of sensitized SRBC upon long-termstorage. Abatch of sensitized SRBC was prepared, preserved at

40C,

and tested for

reac-tivity in the IHA test after various times to

examinethestability of thecells upon storage.

TABLE 3. Antibodyresponsesofpigsexperimentally

exposedtovirulent TGEvirus

Pre-exposurea Postexposure Collection of

Pigno. postexposure

SN IHA SN IHA serum(day)

1 <1 <10 2 10 7

2 <1 <10 4 10 7

3 <1 <10 4 20 7

4 <1 <10 4 40 9

5 <1 <10 4 40 10

6 <1 <10 8 40 10

7 <1 <10 8 40 10

8 <1 <10 8 80 10

9 <1 <10 16 80 10

10 <1 <10 16 80 11

11 <1 <10 16 160 14

12 <1 <10 32 80 14

13 <1 <10 32 80 14

14 <1 <10 128 320 14

15 <1 <10 128 640 14

aSN, SNantibodytiter;IHA,IHAantibodytiter.

TABLE 4. Stability of sensitizedSRBCupon storage

at4°C

Storage IHA antibody titer

period Serum Serum Serum Serum Serum

(days) 1 2 3 4 5

0 160 160 160 320 <10

15 320 160 80 640 <10

30 160 160 80 320 <10

50 160 80 160 160 <10

The results of tests on five sera are shownin

Table 4. Sera used for testing the ability of the sensitized SRBC to measure TGE antibody

werethe samethrough the experiment. No marked changes in antibody titer de-tectedby the IHA test occurred during 50days of storage of the cells at 40C. Furthermore, serum free from SNantibody (serum 5) didnot

agglutinate the sensitized SRBCto anyextent, even at the termination of storage (50 days). Thiswould indicate that sensitized SRBC are

stable upon storageat40C foratleast50days.

DISCUSSION

Although several

serological

tests, such as

the SN (8), bentonite

agglutination

(14),

com-plement fixation (15), and agar

immunodiffu-sion (2, 15), have been

developed by

the pre-vious workers for detecting TGE antibody, they possess certain

disadvantages

for diagnos-tic use.

The SN test is a sensitive and reliable method forserodiagnosis of TGE; however, it is

time consuming and requires facilities for cell culture.

The bentoniteagglutinationtestreported by Sibinovic et al. (14), in which TGE

antibody

agglutinates bentonite

particles

coated with TGEvirus, maybe oflimited value for

diagno-sis of TGE since results do not correlate with those

obtained

inthe SNtest (3).

The complement fixation and

immunodiffu-sion tests are limited in their

sensitivity

for detecting TGE

antibody. Only

serathatpossess

high SN

antibody,

i.e.,

hyperimmune

sera, give positive

results,

whereas sera obtained from TGE convalescentpigsusually show

nega-tivereactionsinbothtests(15).

Bohac and Derbyshire (1, 2) have found that

certain antigens demonstrable by the

immu-nodiffusion test are present indigestive tracts

of pigsinfected with TGE virus, and that TGE convalescent sera can react with those

anti-gens. However, results do not correlate with

thoseobtained in the SN test forsome sera (2). Therefore, the IHA test was explored as a

more rapid, sensitive, and reliable method for detecting and measuring TGE antibody.

TGEantibodywassuccessfullydemonstrated

by the IHA test, using SRBC coated with

highly purified and concentrated TGE virus. Thisindicates that viral particles are the anti-gensreactive inthe IHA test.

Although antibody titers determined by the

IHAtest were approximatelyfivetimes higher

than those determined by the SN test, there

was good correlation (r = +0.865, P < 0.001)

betweentitersobtainedinthetwotests.

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lescentseracollected from pigsexperimentally

exposedtovirulent TGE viruswereallpositive

for IHA antibodyaswell asSNantibody.

Fur-ther, sensitized SRBC used were prepared by

coatingSRBC with highly purified virus. Thus, itappearsthat the IHAtestis specific andmore

sensitive than the SN test for detecting TGE antibody; this suggests that the IHAtest may

be of value for serodiagnosis of TGE.

An antigenic relationship between TGE

vi-rus and hemagglutinating encephalomyelitis

virus, another coronavirus, has been demon-strated by the immunodiffusiontest (11). Sera examined in thisstudywerenottestedfor

anti-body against hemagglutinating encephalomye-litis virus, and it remains to be established whether hemagglutinating encephalomyelitis antibody agglutinates SRBC sensitized with TGE virus.

Sensitized SRBCwerestableuponstorage at

4°C foratleast 50days, which is advantageous inusingthe IHAtestfordiagnosticpurposes.

During these studies, it was noticed that some sera negative for SN antibody

agglutin-ated SRBC sensitized with crude or partially

purified virus. Distinct and specific hemagglu-tination occurred only when SRBCwere

sensi-tized with highly purified virus. Further, more

consistent antibody titerswere obtained when

SRBC weresensitizedwithvirustiteringmore

than 108.4

TCIDs5/ml.

These findings indicate that SRBC should be sensitized with purified and high-titered virus to obtain reproducible results.

Inthe experiment shown inTable 1, 3ml of purified virus, with a titer of 109.1 TCID50/ml, was obtained from 5,000 ml of infected cell

culturefluid, and therecoveryofinfectious

vi-rus was only 15%. The reason for the loss of infectious virus during the purification and concentration procedures is not apparent. If SRBC are sensitized with virus titering 108.8 TCID50/ml, only 12 ml of sensitized SRBC

sus-pensioncanbepreparedfrom 3 ml ofthe

puri-fied virus of 109.1

TCID,,/ml,

orfrom5,000mlof infected cell culture fluid. Thisseverely limits thepractical application of theIHA test. Meth-ods formassproductionandpurificationof TGE

viralantigenmustbe devisedtopermit applica-tion of the IHAtest toserodiagnosis of TGE. A roller bottle culturesystemformassproduction

of TGE virus is now being used, and virus titeringmorethan108°0TCIDs(/mlcanbeeasily

and

regularly obtained.

Therefore,

the

draw-backof the IHA testmentioned above will prob-ablybeovercome inthenearfuture.

LITERATURE CITED

1. Bohac, J., and J. B. Derbyshire. 1975. The detection of transmissible gastroenteritis viral antigens by im-munodiffusion. Can. J. Comp. Med. 39:67-75. 2. Bohac, J., and J. B. Derbyshire. 1976. The detection of

transmissible gastroenteritis viral antibody by

im-munodiffusion. Can. J.Comp. Med. 40:161-165. 3. Bohl, E. H. 1975. Transmissible gastroenteritis, p.

168-188. In H. W.Dunneand A. D. Leman (ed.),Diseases of swine, 4thed. Iowa State University Press, Ames. 4. Fuccillo, D. A., F. L. Moder, R. G. Traub, S. Hensen, and J. Sever. 1971. Microindirect hemagglutination test forcytomegalovirus. Appl. Microbiol. 21:104-107. 5. Furuuchi, S., Y. Shimizu, and T. Kumagai. 1976. Vac-cinationofnewborn pigs with anattenuated strain of transmissible gastroenteritis virus. Am. J. Vet. Res. 37:1401-1404.

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10. McKenna, J. M., F. Zuschek, and J. W. Frankel. 1958. Anhemagglutination test for titration of antibodies topolioviruses. Proc. Soc. Exp. Biol. Med. 97:160-163. 11. Phillips, J. I. H., S. F. Cartwright, and A. C. Scott. 1971.The sizeand morphology of TGE and vomiting and wasting disease viruses of pigs. Vet. Rec. 88:311-312.

12. Sasahara, J., K. Harada, S. Hayashi, and M. Wata-nabe.1958. Studiesontransmissible gastroenteritis inpigs inJapan. Jpn. J. Vet.Sci. 20:1-7.

13. Shimizu, Y., Y. Isayama, Y. Kawakami, N. Murase, and T. Kawano. 1972. Micro indirect hemagglutina-tion test for detecting antibody against infectious bovine rhinotracheitis virus. Natl. Inst. Anim. HealthQ. 12:1-7.

14. Sibinovic, K. H., M. Ristic, S. Sibinovic, and J. 0.

Alberts. 1966. A bentonite agglutination test for transmissible gastroenteritis of swine. Am. J. Vet. Res. 27:1339-1344.

15. Stone, S. S., L. J. Kemeny, and M. T. Jensen. 1976. Partial characterization of the principal soluble anti-gensassociated with the coronavirus oftransmissible gastroenteritisby complement fixation and immuno-diffusion. Infect.Immun. 13:521-526.